Gheorghe-Doru Roiban

Achieving Regio‐ and Enantioselectivity of P450‐Catalyzed Oxidative CH Activation of Small Functionalized Molecules by Structure‐Guided Directed Evolution

Directed evolution of the monooxygenase P450‐BM3 utilizing iterative saturation mutagenesis at and near the binding site enables a high degree of both regio‐ and enantioselectivity in the oxidative hydroxylation of cyclohexene‐1‐carboxylic acid methyl ester. Wild‐type P450‐BM3 is 84 % regioselective for the allylic 3‐position with 34 % enantioselectivity in favor of the R alcohol. Mutants enabling R selectivity (>95 % ee) or S selectivity (>95 % ee) were evolved, while reducing other oxidation products and thus maximizing regioselectivity to >93 %. Control of the substrate‐to‐enzyme ratio is necessary for obtaining optimal and reproducible enantioselectivities, an observation which is important in future protein engineering of these mono‐oxygenases. An E. coli strain capable of NADPH regeneration was also engineered, simplifying directed evolution of P450 enzymes in general. These synthetic results set the stage for subsequent stereoselective and stereospecific chemical transformations to form more complex compounds, thereby illustrating the viability of combining genetically altered enzymes as catalysts in organic chemistry with traditional chemical methods.

Cytochrome P450 Catalyzed Oxidative Hydroxylation of Achiral Organic Compounds with Simultaneous Creation of Two Chirality Centers in a Single C-H Activation Step

Regio- and stereoselective oxidative hydroxylation of achiral or chiral organic compounds mediated by synthetic reagents, catalysts, or enzymes generally leads to the formation of one new chiral center that appears in the respective enantiomeric or diastereomeric alcohols. By contrast, when subjecting appropriate achiral compounds to this type of C-H activation, the simultaneous creation of two chiral centers with a defined relative and absolute configuration may result, provided that control of the regio-, diastereo-, and enantioselectivity is ensured. The present study demonstrates that such control is possible by using wild type or mutant forms of the monooxygenase cytochrome P450 BM3 as catalysts in the oxidative hydroxylation of methylcyclohexane and seven other monosubstituted cyclohexane derivatives.

Regioselective Orthopalladation of (Z)-2-Aryl-4-Arylidene-5(4H)-Oxazolones: Scope, Kinetico-Mechanistic, and Density Functional Theory Studies of the C–H Bond Activation

Orthopalladated complexes derived from (Z)-2-aryl-4-arylidene-5(4H)-oxazolones have been prepared by reaction of the oxazolone with palladium acetate in acidic medium. The reaction is regioselective, only the ortho C-H bond of the arylidene ring being activated, producing a six-membered ring. The scope and reaction conditions of the orthopalladation are dependent on the acidity of the solvent. In CF(3)CO(2)H a large number of oxazolones can be metalated under mild conditions. As acidity decreases a lesser number of oxazolones can be efficiently palladated and harsher conditions must be used to achieve similar yields. The C-H bond activation in acidic medium agrees with an ambiphilic mechanism, as determined from kinetic measurements at variable temperature and pressure for different oxazolones substituted at the arylidene ring. The mechanism has been confirmed by density functional theory (DFT) calculations, where the formation of the six-membered ring is shown to be favored from both a kinetic and a thermodynamic perspective. In addition, the dependence of the reaction rate on the acidity of the medium has also been accounted for via a fine-tuning between the C-H agostic precoordination and the proton abstraction reaction in the overall process occurring on coordinatively saturated [Pd(κ(N)-oxazolone)(RCO(2)H)(3)](2+).

Glycine Fluoromethylketones as SENP‐Specific Activity Based Probes

We report here the synthesis and biochemical properties of a new peptidyl activity‐based probe 1 for SUMO proteases, SENPs. The activity‐based probe has at its C terminus a glycine‐derived fluoromethylketone moiety as a reactive group designed to target the active‐site cysteine of SENPs. Based on a study of the interactions between SENPs and SUMOs, we introduced further design elements that allow the activity‐based probe to selectively target SENPs at low micromolar to high nanomolar concentrations. Moreover, 1 out‐competes SUMO1 from the reversible SUMO1–SENP1 complex, thus suggesting that 1 and SUMO1 share a common binding site on SENP1.

Biocatalytic Route to Chiral Acyloins: P450-Catalyzed Regio- and Enantioselective α-Hydroxylation of Ketones

P450-BM3 and mutants of this monooxygenase generated by directed evolution are excellent catalysts for the oxidative α-hydroxylation of ketones with formation of chiral acyloins with high regioselectivity (up to 99%) and enantioselectivity (up to 99% ee). This constitutes a new route to a class of chiral compounds that are useful intermediates in the synthesis of many kinds of biologically active compounds.

Metal Ion Mediated Self-Assembly Directed Formation of Protein Arrays

Self-assembled inorganic-protein arrays with well-defined and controllable size and structure were obtained through the Fe(II) complexation of protein-conjugated terpyridine units (ligand). The atom-level control of the ligand is obtained through residue-specific conjugation between the complexing unit (terpy) containing an activity-based probe and a corresponding active enzyme (papain). The Fe(II)-based self-assembly performed on this unique building block (ligand) leads to chemical species of unprecedented constitution. The first example presented herein opens the way to a shape and size regime usually reserved to polymers.

Efficient Biocatalytic Reductive Aminations by Extending the Imine Reductase Toolbox

Chiral secondary and tertiary amines are ubiquitous in pharmaceutical, fine, and specialty chemicals, but their synthesis typically suffers from significant sustainability and selectivity challenges. Biocatalytic alternatives, such as enzyme‐catalyzed reductive amination, offer several advantages over traditional chemistry, but industrial applicability has not yet been demonstrated. Herein, we report the use of cell lysates expressing imine reductases operating at 1:1 stoichiometry for a variety of amines and carbonyls. A collection of biocatalysts with diversity in coverage of small molecules and direct industrial applicability is presented.

Reactivity of Unsaturated 5(4H)-Oxazolones with Hg(II) Acetate: Synthesis of Methyl N-Benzoylamino-3-arylacrylates

Abstract An efficient and high-yield procedure to prepare methyl N-benzoylamino-3-arylacrylates from unsaturated (Z)-2-aryl-4-arylidene-5-(4H)-oxazolones and Hg(OAc)2 in methanol is described herein. The observed reactivity of mercury(II) acetate here is different than its usual metallating behavior and it cleaves the unsaturated oxazolone ring without change of stereochemistry. GRAPHICAL ABSTRACT

A general solid phase method for the synthesis of sequence independent peptidyl-fluoromethyl ketones.

We present here a new, general, solid phase strategy for the synthesis of sequence independent peptidyl-fluoromethyl ketones using standard Fmoc peptide chemistry. Our method is based on the synthesis of bifunctional linkers which allows the incorporation of amino acid fluoromethyl ketone unit at the C-terminal end of peptide sequences. Application of this approach for the synthesis of activity based probes for SENPs is also described.

CHAPTER 9:GSK: Biocatalyst Discovery and Optimisation

With the world’s increasing demand for access to medicines, GSK continuously seeks new technology solutions to enable more efficient production of drug molecules and respond to the pressure to quickly deliver high quality medicines at lower cost. Not only is the focus on reducing the time it takes to bring a drug to market, but doing so with a focus on sustainable and cost efficient manufacturing techniques. Biocatalysis and synthetic biology are key technologies pursued at GSK as a result of its potential to deliver environmentally friendly synthesis of small molecules and the provision of complementary access to active compounds, alongside the more traditional synthetic chemistry approaches. Significant effort has been put into building in-house enzyme panels which have been assembled based on bioinformatic analysis. Panels comprising different enzymatic classes represent a ready-to-use platform for rapid screening of activity and enantioselectivity for the synthesis of small, chiral API building blocks. Optimisation of hits identified through screening, through use of in-house capabilities, ensures fast implementation of an enzymatic step into a manufacturing route or to support research demands.